Project Summary Proliferative diabetic retinopathy (PDR) is a leading cause of blindness in adults, mostly due to neovascularization in ischemic and hypoxic retinal tissues. While monthly injection of anti-VEGF provides relief, it is associated with major side effect such as retinal detachment, and over a third of the patients are refractory to the antibody treatment. The long term objective of this project is to investigate how the endothelial oxygen sensing mechanism regulates retinal angiogenesis, and develop novel strategies to curb neovascularization in ischemic retinal tissues. Typically, hypoxia drives angiogenesis by paracrine mechanisms wherein hypoxia- inducible factor-? proteins upregulate the expression of angiogenic factors, which activate their cognate receptors on nearby endothelial cells (EC). In well oxygenated tissues, HIF-? proteins degrade rapidly following oxygen dependent prolyl hydroxylation by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). Thus, deficiency in PHDs mimics hypoxia at the molecular level by allowing HIF-? proteins to accumulate. Indeed, our published studies demonstrate that post-natal PHD2 deficiency globally or in non- endothelial cells promotes vascular growth or survival. In recent Preliminary Studies, we made a rather unexpected observation that EC specific PHD2 deficiency (Phd2EC-/-) inhibited instead of promoting retinal vascular development, despite high level accumulation of HIF-1? and HIF-2?. On the other hand, Flt-1 was upregulated, which is known to sequester VEGF-A. In the mouse model of oxygen-induced retinopathy (OIR), a proxy of PDR as well as ROP (retinopathy of prematurity), Phd2EC-/- mice also displayed greatly reduced neovascularization. These findings led to our Central Hypothesis stating that retinal angiogenesis is dynamically regulated by two opposing oxygen sensing mechanisms: in the cells of the retinal parenchyma, hypoxia activates angiogenesis by HIF- dependent expression of angiogenic factors; whereas in the retinal ECs, hypoxia inhibits angiogenesis by upregulating anti-angiogenic molecules such as Flt-1. Aim 1. Investigate how the endothelial oxygen sensing mechanism regulates retinal vascular development. Examine whether EC specific deletion of HIF-1?, HIF-2?, or Flt-1 rescues retinal vascular development in Phd2EC-/- mice; whether the accumulation of oxygen resistant but otherwise normal HIF-1? and HIF-2? suppresses angiogenesis; and whether HIF-1? and HIF-2? directly interact with Flt-1 regulatory elements; Aim 2. Investigate whether the oxygen sensing mechanisms regulating developmental angiogenesis also operate during ischemic neovascularization in the OIR model. Aim 3. Test lentiviral vector-mediated anti-angiogenesis strategies. Lentiviral vectors will be constructed that express PHD siRNAs or membrane - anchored truncated Flt-1 (tFlt-1), all spatially restricted to retinal ECs and likely further restricted to ECs in ischemic retinal locations. The ability of these vectors to suppress ischemic retinal neovascularization will be evaluated in the OIR model. These studies may potentially lead to an innovative therapy to suppress neovascularization associated with PDR.